Article pubs.acs.org/Langmuir
On-Demand Droplet Fusion: A Strategy for Stimulus-Responsive Biosensing in Solution Praveena Mohan, Patrick S. Noonan, Matthew A. Nakatsuka, and Andrew P. Goodwin* Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Ave., 596 UCB, Boulder, Colorado 80309, United States S Supporting Information *
ABSTRACT: A novel strategy is reported for biochemically controlled fusion of oil-in-water (O/W) droplets as an in-solution sensor for biological targets. Inspired by the SNARE complex in cells, the emulsions were stabilized by a combination of phospholipids, phospholipid−poly(ethylene glycol) conjugates, and cholesterol-anchored oligonucleotides. Prior to oligonucleotide binding, the droplets were stable in aqueous media, but hybridization of the oligonucleotides in a zipperlike fashion was shown to initiate droplet fusion. Using image analysis of content mixing of dye-loaded droplets, fusion specificity was studied and optimized as a function of interfacial chemistry. Changing the orientation of the anchored oligonucleotides, using long-chain phospholipids (C18 and C22), and binding a complementary oligonucleotide slowed or even halted fusion completely. Based on these studies, a sensor for the biomarker thrombin was designed using competitive binding of aptamer strands, with droplet fusion increasing as a function of thrombin addition in accordance with a simple binding model, with sensitivity down to 100 nM and with results in as little as 15 min. Future efforts will focus on utilizing this mechanism of content mixing to facilitate highly sensitive detection via modalities such as magnetoresistance or chemiluminescence.
1. INTRODUCTION The sequestration of molecules and other contents into dispersible colloids is of widespread importance for applications in biotechnology, medicine, and analytics. Confinement of encapsulated contents into droplets allows the dispersal of many different types of molecules in suspension while maintaining their mutual separation. If the droplets do fuse, or coalesce, their contents will be mixed together but still remain separate from other molecules in suspension, allowing selective interaction in a confined volume. While oil droplets dispersed in aqueous media spontaneously fuse to reduce their overall surface area and energy, the use of specific binding events to control droplet fusion is less trivial. Such techniques have been developed for microfluidics,1−4 in which the flow and channel design help to control the spatial location of each droplet. While microfluidics have been quite successful in analytics, the technique is limited to small volumes and flow rates. A technique that could fuse specific oil-in-water (O/W) droplets together rapidly and in bulk would help to pave the way for in-solution detection of biologics without any washing steps. In this paper, we present a new methodology for rapid and controlled mixing of specific droplets in bulk suspension. This method was inspired by the N-ethyl-maleimide-sensitive-factor attachment protein (SNARE), which induces fusion between cell organelles and membranes. The key step in the SNAREmediated fusion process is the formation of a four-helix bundle, the formation of which applies a directional force that draws the vesicle and the membrane close to one another and overcomes © 2014 American Chemical Society
the entropy loss and steric hindrance associated with lipid mixing.5,6 This in turn facilitates pore formation between the two membranes, followed by completion of the fusion process. Synthetic mimics of this “zippering process” have been designed in liposome models using simple peptide constructs,7 peptide-antigen interactions,8 and oligonucleotides.9,10 In particular, Höök and coworkers employed DNA−cholesterol conjugates anchored in the outer leaflet as associative groups for initiating liposome fusion.11−13 In applying such technologies to O/W emulsions, it was important to demonstrate that the formulations used for liposome bilayers can be adapted to monolayers at the surface of a droplet. Also, the fusion process itself is thermodynamically favored through the consequential reduction in interfacial energy, so additional steric barriers must be added to the monolayer to prevent nonspecific coalescence under normal conditions. In our work with microbubbles and liposomes,14−17 as well as others’ with bubbles, emulsions, and liquid crystal monolayers,18−23 monolayers consisting of phospholipids and conjugates of poly(ethylene glycol) and 1,2-distearoyl-snglycero-3-phosphoethanolamine (DSPE-PEG) have proven successful in maintaining stable colloidal suspensions. However, steric barriers are expected to retard specific fusion as well. Thus, careful utilization of both PEG length and DSPE-PEG Received: June 24, 2014 Revised: September 9, 2014 Published: September 27, 2014 12321
dx.doi.org/10.1021/la502483u | Langmuir 2014, 30, 12321−12327
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2.5. Image Analysis: Measurement of Fusion and Size Distribution. ImageJ (NIH) was used to select one green and one red channel image from the same focus among the Z-stacks and save them separately in a tif format.24 A MATLAB code (MATLAB and Statistics Toolbox Release 2013a, The MathWorks, Inc., Natick, MA) was written to select droplets found in both channels and then measure the mean intensities and surface area for each droplet. The mixing of red and green dyes was quantified by determining intensity ratio, IG/(IG + IR); thus, a completely red droplet would have a value of 0 and a completely green droplet would have a value of 1. A histogram of intensity ratio versus count was plotted in bin increments of 0.05, and a ratio of summed droplet counts between 0.15 and 0.85 was compared to the total droplet count (see Figure S2, Supporting Information). 2.6. Detection of Thrombin Using Thrombin Aptamer. Emulsions were prepared as above, but during the vesicle preparation step, thrombin aptamer and 5′Chol-DNA were added to the lipid stock and DSPE-PEG solution. This sample was then stirred at 75 °C for 30 min. The formulation contained 1.3 mM DPPC, 40 μM DSPEPEG2000, 1.0 μM thrombin aptamer, and 0.65 μM Link-1 DNA. The molar ratio of DNA/PEG/lipid was 1:62:2000. Link-2 incorporated vesicles were prepared as before. Again, green and red 5CB emulsions were made with Link-1/TA vesicles and Link-2 vesicles. The fusion experiments were repeated by mixing equal amounts of green and red emulsions with different thrombin concentrations of 0, 50, 100, 200, 400, and 800 nM. The emulsion fusion was imaged at 45 min after mixing, and then processed to report the maximum fusion efficacy over the range of thrombin concentrations.
density are necessary for maximizing emulsion fusion specificity.
2. EXPERIMENTAL SECTION 2.1. Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2dibehenoyl-sn-glycero-3-phosphocholine (DBPC); and 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (ammonium salt) (DSPE-PEG) (PEG molecular weights of 1000, 2000, and 5000) were obtained from Avanti Polar Lipids (Alabaster, AL). 4-Cyano-4′-pentylbiphenyl (5CB) and 9,10-bis(phenylethynyl)anthracene (BPEA) dye were obtained from TCI America (Portland, OR). Cholesterol-modified DNA sequences (Chol-DNA) and thrombin aptamer (TA) were obtained from Integrated DNA Technologies (Coralville, IA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dyes were obtained from Sigma-Aldrich (St. Louis, MO). Chloroform, Tris base, and sodium chloride (NaCl) were obtained from Fisher Scientific (Pittsburgh, PA). 2.2. Preparation of Lipid Stock Solution. Tris buffer of 10 mM Tris base and 100 mM NaCl was prepared, and its pH was adjusted to 8.0 with 1 M HCl. A total of 16 mg of lipid (DPPC, DSPC, or DBPC) was dissolved in 1.5 mL of chloroform and sonicated in a water bath at 32 °C for 2 min until the solution was clear. The solvent was evaporated in a 100 mL round-bottom flask under vacuum in a rotary evaporator for 30 min. Once the chloroform was evaporated and the film was formed, a stock solution of 4 mg/mL lipid was prepared by hydrating the film in 4 mL of Tris buffer. The mixtures were stirred for ca. 40 min in a heated water bath at 75 °C for DPPC and DSPC; and at 90 °C for DBPC. 2.3. Preparation of Chol-DNA Loaded Vesicles and 5CB Emulsions. The lipid/PEG/DNA mixtures were prepared by adding Chol-DNA and DSPE-PEG in aqueous media to the lipid stock solution. The concentration of lipid in the final formulation was 1.3 mM; and Chol-DNA and DSPE-PEG concentrations were adjusted according to the experiments. This mixture was then heated to 75 °C for DPPC and DSPC and at 90 °C for DBPC and stirred for 30 min. Once the solutions were cooled to room temperature, 2 v/v % 5CB was added to the mixtures. The samples were then emulsified using a 40 kHz probe sonicator (Branson SLPe, Branson Ultrasonics, Danbury, CT) with an output power of 120 W. The samples were then centrifuged at 1600g for 1 min to remove large and nonencapsulated 5CB droplets. The supernatants were centrifuged again at 6000g for 4 min to pellet down the encapsulated 5CB droplets. The supernatant containing excess lipids and smaller droplets (
